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Research articles

ScienceAsia 39(2013): 596-604 |doi: 10.2306/scienceasia1513-1874.2013.39.596


Characterization of a cDNA encoding cystatin with antifungal activity from Siam tulip Curcuma alismatifolia


Ruangwit Porruana, Ruttaporn Chundetb, Somboon Anuntalabhochaia,c,*

 
ABSTRACT:     Cystatins are cysteine protease inhibitors involved in defence mechanisms against pests and pathogens. Here, the cystatin CaCPI gene was isolated from a cDNA library of the Siam tulip (Curcuma alismatifolia cv. Chiang Mai Pink). The full length cDNA of 601 bp contains 372 bp of an open reading frame encoding 123 amino acids flanked by 5′and 3′ untranslated regions of 32 and 197 bp, respectively. The deduced amino acid sequence consists of a putative N-terminal secretory signal peptide of 22 amino acids and an estimated molecular mass of 11.2 kDa of the mature protein. The CaCPI protein contains all of the highly conserved blocks included Gly31-Gly32, the reactive site motif QXVXG (Q76V77V78 A79G80), the P106-W107, and the [LVI]-[AGT]-[RKE]-[FY]-[AS]-[VI]-X-[EDQV]-[HYFQ]-N (L49G50R51 F52A53V54 D55Q56 H57N58) block that is common among plant cystatins. The CaCPI gene was cloned into a pDEST17 expression vector and was then transformed into Escherichia coli strain BL21-Star to produce a recombinant CaCPI protein. After induction with 1 mM IPTG, the cell lysate of E. coli-carrying pDEST17-CaCPI generated a CaCPI protein about 12 kDa in size as measured using SDS-PAGE. Pre-incubation of the 5–30 µM CaCPI protein samples with 5 µM papain is known to decrease papain activity. Antifungal activities of the purified recombinant CaCPI protein against phytopathogenic fungi were tested. The CaCPI protein could suppress mycelium growth of Fusarium oxysporum, Colletotrichum capsici, and Pyricularia grisea phytopathogenic fungi.

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a Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 Thailand
b Division of Biotechnology, Faculty of Science, Maejo University, Chiang Mai 50290 Thailand
c Biotechnology Unit, University of Phayao, Phayao 56000 Thailand

* Corresponding author, E-mail: soanu.1@gmail.com

Received 20 Jan 2013, Accepted 11 Jul 2013